1v04



Serum Paraoxonase-1 (PON1) via directed evolution

Overview
Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate detoxification and in prevention of atherosclerosis. There are three members in this family, PON1, PON2 and PON3, which share 60-70% nucleic acid identity. The most studied enzymes are the two isoenzymes of PON1, which differ in the residue at position 192 (Q/R). The primary activity of PON1 is lactone hydrolysis; however, this enzyme has many other activities. One of the interesting activities is the hydrolysis of organophosphates. PON1 can catalyze a variety of nerve agents such as cyclosarin, soman, etc. Therefore, it is aimed to be a nerve agent scavenger. In addition, PON1 has a role in prevention of atherosclerosis and is found to be attached to the high-density lipoprotein (HDL, “good cholesterol”). Human PON1 is not stable, and tends to aggregate in the absence of detergents. In addition, it cannot be expressed in bacteria or yeast for protein over-expression, mutagenesis and protein engineering. Therefore, this protein was submitted to directed evolution in order to over-express it in E.coli and to increase its solubility. Family shuffling of four PON1 genes (human, mouse, rabbit and rat) resulted in many variants that could be expressed in E.coli, but only one of them (G2E6-variant) led to quality-diffracted crystals. The recombinant-PON1 (rePON1) G2E6 variant, exhibits 91% homology to the wt rabbit PON1 and 86% homology to the human PON1. This variant exhibits resemble catalytic activity to human PON1.

Structural features


The crystal structure of rePON1 shows  a six-bladed  β-propeller fold. PON1 has a unique addition to the β-propeller scaffold: three  α-helixes, which are located on the top of the propeller. These helixes are likely to be involved in the anchoring to the HDL particles. In addition, a stabilizing disulfide bridge between Cys-42 and Cys-353 was found. The structure of rePON1 resembles that of Loligo vulgaris DFPase (PDB 1e1a). Both are six-bladed propellers with each blade consisting of four β-sheets. Moreover, in both structures two calcium ions can be found in their central tunnel. The calcium atom, which resides at the top of the tunnel, is assigned as the ‘catalytic calcium’ (Ca-1), whereas the other calcium at the central section is assigned as the ‘structural calcium’ (Ca-2). The latter is involved in stabilization of the structure. In addition to the two calciums, there is a phosphate ion, which is bound to Ca-1 in the active site. This phosphate ion is thought to come from the mother liquor. The Loligo vulgaris structure lacks the three α-helixes found in the rePON1 structure. The catalysis mechanism of organophosphates has yet not been discovered. However, determination of the pH-rate profile of rePON1 proposed participation of a Histidine (His) dyad in the lactonase activity of PON1. In hydrolytic enzymes, His often serves as a base, deprotonating a water molecule, and thus generating the attacking hydroxide ion that produces hydrolysis. The His-dyad (His-115 and His-134) resides near both Ca-1 and the phosphate ion. The hypothesis is that His-115 acts as a general base to deprotonate a single water molecule, thus generating the attacking hydroxide, while His-134 acts in a proton shuttle mechanism to increase His-115’s basicity. In addition, His-115 was found to have distorted dihedral angles, thing that characterizes many catalytic residues. This observation was supported by site mutation of both His-115 and His-134, which result in a dramatic decrease in both arylesterase and lactonase activity of PON1. Interestingly, the organophosphate hydrolysis activity of these mutations was not affected. Therefore, different loactions in the rePON1 binding site are postulated to have different enzymatic activities in its  overall structure.